1. Field of the Invention
This invention relates to a scanning optical microscope, and more particularly to a scanning optical microscope having a confocal optical system by which an object can be scanned at a high speed.
2. Description of the Prior Art
In a scanning optical microscope, light from a light source such as a laser is focused as a minute light spot on an object by an objective lens, and the object is scanned by the light spot to obtain an image of the object. Compared with conventional optical microscopes, scanning optical microscopes provide images of high contrast since no diffused light comes from the area other than the light spot. Further, special microscopy such as confocal microscopy, differential phase microscopy, etc., can be done easily by scanning optical microscopes, and it is also possible to visualize various physical phenomena which cannot be observed by means of conventional optical microscopes, such as OBIC (optical beam induced current) images, photo-acoustic images, etc. Therefore, scanning optical microscopes are expected to be useful microscopes in the semiconductor and material industries as well as biology and medical science.
Many of the conventional scanning optical microscopes scan an object by moving in a horizontal direction a stage on which the object is mounted, without shifting the position of a light spot, or by providing a reflecting mirror such as a polygonal rotating mirror, a galvanometer mirror, etc., in the optical path from a light source to an objective lens to shift in a horizontal direction a light spot formed on an object. However, since these scanning systems cannot come up with the horizontal scanning of a television system because of their low scanning speed, real-time observation of an object is impossible. In order to resolve such problem, the inventor of the present invention has proposed in a laid-open Japanese patent application, publication No. 61-219919, a scanning optical microscope in which an acousto-optic light deflector (hereinafter AOD, if applicable) is used instead of the above-mentioned mirror so that the scanning speed can be higher and real-time observation of an object can be made.
FIG. 1 shows an optical system of a scanning optical microscope disclosed in the laid-open Japanese patent application, publication No. 61-219919. The optical system comprises a beam splitter 1, a first light deflector 2 formed by an AOD, pupil transfer lenses 3 and 4, a second light deflector 5, a pupil projection lens 6, an imaging lens 7, and an objective lens 8. Numeral 9 denotes a pupil of the objective lens 8 and numeral 10 denotes an object or specimen. The second light deflector 5 is arranged in the position conjugate with the pupil 9 of the objective 8 with respect to the pupil projection lens 6 and the imaging lens 7, and the first light deflector 2 is located in the position conjugate with the second light deflector 5 with respect to the pupil transfer lenses 3 and 4. The first light deflector 2 performs horizontal scanning while the second light deflector 5 effectuates vertical scanning. Further, a collector lens 11, a pinhole 12 and a detector 13 are arranged.
A beam 14 from a light source (not shown) such as a laser passes through the beam splitter 1 and enters the first light deflector 2. The light exiting from the first light deflector 2 varies in its exit angle from the most deflected position shown by the dotted lines through the non-deflected position shown by the full lines to the most deflected position on the opposite side (not shown). The beam 14 passes through the pupil transfer lenses 3 and 4 and enters the second light deflector 5 where the exit angle of light varies in the same manner as in the first light deflector 2. The beam 14 deflected two-dimensionally by both light deflectors 2 and 5 is caused to enter the pupil 9 of the objective 8 by the pupil projection lens 6 and the imaging lens 7. Further, the beam 14 is focused to its diffraction limit and scans the specimen 10 two-dimensionally. The light reflected from the specimen 10 returns through the objective 8, the imaging lens 7, the pupil projection lens 6, the second light deflector 5, the pupil transfer lenses 4 and 3, and the first light deflector 2. The returned reflected light is taken out by the beam splitter 1 and becomes a detection beam 17. Since the detection beam 17 has passed the light deflectors 5 and 2 again, it returns to the same position. The detection beam 17 is focused by the collector lens 11 and detected by the detector 13 through the pinhole 12. Thus, an image of high resolution by reflected light can be obtained.
In addition to high resolution of an image, this method using a pinhole has an important feature that a sliced image of an object can be obtained as described below.
FIG. 2 illustrates a principle of obtaining a sliced image of an object by reflected light when a pinhole is used, that is, the principle of confocal microscopy. For the purpose of simplification, the scanning optical system is omitted. There are shown a point light source 21, a beam splitter 22, an objective lens 23, a specimen 24, a pinhole 25, and a detector 26. The pinhole 25 is located in a position conjugate with the point light source 21, that is, an image of the point light source 21 is formed on a plane 27 in the specimen 24 by the objective 23, and the image is formed again at the pinhole 25 by the same objective 23. Therefore, the above-described system is called a confocal system.
Light from the point light source 21 enters the objective 23 and illuminates a point in the plane 27 in the specimen 24. Reflected light becomes a beam 29 which is reflected by the beam splitter 22, passes through the pinhole 25 and is detected by a detector 26. A light beam 30 reflected from another plane 28 (located out of focus) in the specimen 24 has an expansion at the pinhole 25 and therefore hardly reaches the detector 26. Thus, since light other than that from the plane 27 including the point illuminated by the point light source 21 is not detected, a sliced image of a thick specimen can be easily obtained.
In reality, a light scanning system as shown in FIG. 1 is inserted between the point light source 21 and the objective lens 23. Since the pinhole 25 is minute and cannot be moved in synchronism with scanning, the pinhole 25 must be positioned on the same side as the light source 21 with respect to the light scanning system.
In the above, confocal microscopy by using light reflected from an object is described. However, if the same scanning optical microscope is used for confocal fluorescence microscopy, the following problems arise:
An AOD is a device for deflecting light through diffraction grating produced by a sound wave. The deflection angle .theta., i.e., the angle between a light beam incident on an AOD and a light beam exiting from the AOD is given by the following formula: ##EQU1## where
.lambda. is the wavelength of the light incident on the AOD;
v is the sound velocity in the AOD; and
f is the frequency of a sound wave applied to the AOD. Therefore, the deflection angles .theta..sub.L and .theta..sub.F of a laser beam of wavelength .lambda..sub.L projected on an object and a fluorescent beam of wavelength .lambda..sub.F emitted from the object, respectively are given by the following formulas: ##EQU2## Thus, even if the same sound wave is applied to the same AOD, the deflection angles differ from each other. Consequently, when an AOD intervenes, traveling directions of a laser beam and a fluorescent beam are different from each other, so that both beams do not come to the same position. Therefore, the pinhole 12 in FIG. 1 must be shifted slightly in a direction perpendicular to the optical axis, depending upon which is used for observing an object, a laser beam or a fluorescent beam. Moreover, the difference between the deflection angles of a laser beam and a fluorescent beam, represented by the following formula: ##EQU3## varies with the frequency f of a sound wave. Thus, when the deflection angle is varied by varying the value f in order to scan an object, the amount of discrepancy between the traveling directions of the laser beam and the fluorescent beam varies. Strictly speaking, unless the position of the pinhole 12 in FIG. 1 is minutely adjusted in accordance with the variation of the deflection angle, fluorescent light of wavelength .lambda..sub.F goes out of the pinhole 12 (instead, fluorescent light having a slightly different wavelength passes through the pinhole 12), so that an accurate fluorescence microscopy cannot be realized.
Moreover, the diffraction efficiency of an AOD is dependent on the wavelength of incident light. When an AOD having a high diffraction efficiency for laser beams is used, there is a problem that the diffraction efficiency for fluorescent beams is low and the intensity of fluorescent light is reduced.
As described above, in a scanning optical microscope disclosed in the laid-open Japanese patent application, publication No. 61-219919, the detection of a sliced image of a specimen by using fluorescent light can be realized in principle by a detection method using a pinhole. However, since the detection is performed through an AOD, the wavelength of detected fluorescent light varies and the diffraction efficiency cannot be improved. Thus, this scanning optical microscope is not practical.